In Vivo Longitudinal Mapping of Brain Iron Accumulation After Pilocarpine-Induced Status Epilepticus

Abstract

Iron accumulations have been identified in resected tissue from patients with refractory temporal lobe epilepsy. These deposits are linked to ferroptosis, a form of nonapoptotic cell death in which iron catalyzes the formation of reactive oxygen species, leading to lipid peroxidation. Experimentally, this process has recently been associated with seizures based on the increased levels of specific markers (4-hydroxynonenal and malondialdehyde) in the brain and plasma. Quantitative susceptibility mapping (QSM) offers an opportunity to detect the iron accumulations in vivo. In this study, we investigated how pilocarpine-induced status epilepticus contributes to the generation of iron deposits in diverse cerebral regions and whether QSM can detect these deposits longitudinally. We scanned 14 animals (n = 10 experimental and n = 4 control) at five different time points (pre-status epilepticus induction and 1, 7, 14, 21 days postinduction) using QSM. We identified iron deposits in the caudate putamen, hippocampus, thalamus, and primary somatosensory cortex of experimental animals, which is consistent with histological findings. The initial size of the hippocampal iron deposits significantly increased over the following weeks. None of these effects was observed in the control animals. The presence of cerebral iron depositions in epilepsy-related brain structures suggests that they could be involved in the onset, development, and progression of spontaneous recurrent seizures. Furthermore, noninvasive, longitudinal in vivo mapping of brain iron deposits could be a potential imaging marker in neurological disorders such as epilepsy. Future experiments will be required to determine the origin of the iron and avoid its progressive accumulation.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s12035-026-06060-y.

Introduction

The biological relevance of iron lies in its ability to donate electrons, playing a crucial role in diverse cellular processes such as DNA replication, the electron transport chain, and energy production. However, the reaction between iron and molecular oxygen can generate toxic compounds, known as reactive oxygen species (ROS) [1]. The abnormal accumulation of iron in the brain can trigger inflammation, neurotransmitter oxidation, neuronal communication failure, myelin sheath degeneration, astrocyte dysregulation, and cell death [2]. Features that have been observed in diverse neurological disorders or neurodegenerative processes such as stroke [3], traumatic brain injury [4], neuroinflammation [5], Alzheimer’s disease [6], Parkinson’s disease [7], and Huntington’s disease [8]. Other studies have reported that stress-induced iron accumulations can also lead to the generation of hemosiderin, a water-insoluble iron storage protein detectable by Perls’ staining [9, 10]. The cerebral accumulation of this protein appears in subarachnoid hemorrhage [11, 12], traumatic brain injury [13], high-altitude cerebral edema [14], and microbleeds secondary to recurrent ischemic strokes or status epilepticus [15, 16]. Interestingly, high chances of developing epilepsy have been reported in these cases. Moreover, hemosiderin deposits are also found surrounding brain tumors or cerebral cavernous malformations [17, 18]; therefore, an incorrect or partial removal may induce seizures [19, 20].

During the last five years, it has been suggested that an imbalanced iron metabolism can contribute to ferroptosis, a process possibly involved in the pathophysiology of epilepsy [21, 22]. The term ferroptosis was coined in 2012 to describe a form of nonapoptotic cell death in which iron, via the Fenton reaction, catalyzes ROS and promotes membrane damage as a consequence of massive lipid peroxidation [23]. In contrast to apoptosis and necrosis, ferroptosis has its own morphological, biochemical, and genetic features. These are mainly characterized by mild chromatin condensation, cytoplasm and cytosolic organelle swelling, plasma membrane integrity deterioration, and mitochondrial alterations [23]. Diverse studies have also reported that tissues undergoing ferroptosis show increased levels of specific biomarkers, including 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) [24–27].

Experimental studies have shown increased levels of 4-HNE, MDA, and cyclooxygenase-2 (COX-2) during epilepsy development [28–30]. Altered antioxidant systems will induce an increase in the expression of the cystine/glutamate antiporter system (x_c-) [31–34] and a decrease in the concentration of glutathione and glutathione peroxidase. Other authors have described that inhibiting ferroptosis could attenuate seizures in mice [35, 36] or even exert neuroprotective effects [37]. Despite the recent evidence, the dynamics of cerebral iron deposition under convulsive conditions has not yet been fully elucidated.

Quantitative susceptibility mapping (QSM), an advanced magnetic resonance imaging technique, has provided noninvasive insight into tissue susceptibility to iron content variations [38–40]. Aggarwal and collaborators [38] were the first to describe iron accumulations in specific brain regions after status epilepticus induction by using pilocarpine-treated rats. Later clinical or experimental imaging findings were further validated by histological or immunohistochemical techniques, confirming the involvement of ferroptosis in epilepsy [41]. The aim of this study was to longitudinally describe the dynamics of brain iron accumulations caused by pilocarpine-induced status epilepticus in rats. We used QSM to longitudinally detect and track the total susceptibility of the iron deposits in vivo. Next, imaging findings were compared to histological assessments of iron deposits in specific staining preparations from the same animals. Perls’ staining has long been used to macroscopically examine the distribution of iron in the brain. We performed this technique, enhanced by diaminobenzidine (DAB), to visualize iron deposits as hemosiderin accumulations (yellow-brown granules made of partially degraded ferritin and lysosomes) [9, 10].

Methods

Animals

Adult male Sprague–Dawley rats were provided by the Institute of Neurobiology’s animal facility. They were maintained under a 12-h light/dark cycle with access to food and water ad libitum. All the rats were acclimatized to the room conditions (20–22 °C, 50–60% humidity) for at least one week before any experimental manipulation. The protocol was approved by the Institute of Neurobiology Ethics Animal Care Committee (protocol 105 A). The procedures were performed in accordance with the Mexican Federal regulations for animal experimentation (NOM-602-ZOO-1999) and ARRIVE guidelines [42]. The number of animals was kept as small as possible.

Intracerebral Injection of Iron Chloride

Five healthy rats (60 days old) were used to evaluate the sensitivity of our QSM protocol for detecting brain iron deposits. Animals were anesthetized with an intraperitoneal injection of ketamine/xylazine mixture (70 and 10 mg/kg, respectively) and placed in a stereotactic frame. An incision was made midline along the scalp, and the skull was exposed using sterile surgical techniques. Based on the Compact Paxinos and Watson atlas 6th edition [43], a small hole was drilled into the skull at the following coordinates: anteroposterior −5.6 mm, lateral −5.5 mm, and ventral from skull surface 7.4 mm. Then, a 5 µl Hamilton syringe was adapted to the stereotactic frame arm and gradually lowered into the right ventral hippocampus to infuse 2 µl of iron chloride (FeCl₃) solution or vehicle (sterile 0.1 M phosphate-buffered saline [PBS]; Sigma-Aldrich) at a rate of 0.1 µl/min using a WPI Microsyringe Pump Controller. Four rats received different concentrations of FeCl₃ solution (0.1, 1, 10, and 100 mM); the fifth rat received 0.1 M PBS. The wound was sutured, and the animals were transferred back to their cages. All the animals were scanned and perfused the next day. The iron deposits were detected by Perls’ staining (see below).

In Vivo MRI Acquisitions

The acquisition protocol was carried out at the National Laboratory of Magnetic Resonance Imaging using a 7-T Bruker animal scanner interfaced to a Paravision 7.0 console. In vivo QSM imaging was acquired using a transmit–receive volume coil with an inner diameter of 72 mm.

Animals were anesthetized with a 5% sevoflurane/oxygen mix. This concentration was reduced by 40% to maintain anesthesia during image acquisition. The respiration rate was monitored and kept at 40–50 breaths per minute. Body temperature was maintained using a warm water circulation system. Inhaled anesthesia was suspended upon termination of the imaging session. Once the rats were fully recovered, they were transferred back to their polypropylene cages.

Datasets for QSM were acquired using a three-dimensional multiecho gradient sequence with the following parameters: TR = 47.2 ms, TE1 = 1.942 ms, echo spacing = 2.1 ms, 8 echoes, flip angle = 16.5°, and spatial resolution = 200 × 200 × 500 mm³. The scan time was 26 min.

Image Data Processing and Analysis

The initial processing was done using brkraw version 0.3.11 (https://brkraw.github.io), MRtrix 3.0, and FSL. Here, raw data were converted to magnitude and phase maps in NifTI format; cerebral maps were also generated in this format (Fig. 1A). The resulting maps were processed with SEPIA version 1.2.1 (https://sepia-documentation.readthedocs.io) and run in MATLAB 2023a (MathWorks, Natick, MA). Then, we followed the next steps to create the susceptibility maps: 1) combination of multiple echoes using the tool Optimum Weights [44]; 2) phase unwrapping using the Laplacian method implemented in MEDI toolbox [45]; 3) background field removal with the VSHARP method [46] (max radius = 10 voxels, min radius = 3 voxels, residual B1 removal using 4th-order 3D polynomial); and 4) creation of QSMs with the STAR-QSM method [47] (pad size = 12 voxels). Finally, the maps were exported to NifTI (Fig. 1B). For reproducibility purposes, our SEPIA processing configuration file is available in the Supplementary Information.

Fig. 1.

Image processing and analysis. The initial processing consisted of converting the raw data into magnitude and phase maps (A). SEPIA was used as the quantitative susceptibility mapping (QSM) pipeline analysis tool (B). FIJI ImageJ was used for the quantitative analysis of iron deposits identified in the QSM images (C). The red box delineates the zone where the iron deposit was detected (dark hole with a surrounding hyperintensity). For each accumulation, we measured the volume (mm³), the mean susceptibility (ppm), and the total susceptibility (ppm·mm³) (C)

MRtrix 3.0 with MRView visual interface was used to detect iron deposits [48]. Here, we applied a filter on the QSM maps at > 0.1 ppm to identify the paramagnetic intensities. For a precise anatomical localization of the iron deposits, we used the T2-weighted TurboRARE structural images.

FIJI ImageJ was used for the quantitative analysis of the total susceptibility of the iron deposits identified in the QSM images. Each accumulation was treated as a region of interest and duplicated in a substack to cover its entire surface in three dimensions. Then, following Liu et al. [49], we applied a threshold of 0.05 ppm to evaluate only paramagnetic intensities; the area and intensity of each deposit was measured on each slice. The size of the deposits was calculated by multiplying the paramagnetic area of each slice (mm²) by the slice thickness (0.5 mm). The resulting value (mm³) of each slice, when added, provides an approximate value of the deposit’s volume. Finally, by multiplying this volume by the mean voxel value (χ̄·V), we obtained the deposit’s total susceptibility (ppm·mm³) (Fig. 1C).

Pilocarpine-Induced Status Epilepticus

Once we demonstrated that iron deposits can be detected by our QSM protocol, we proceeded to the next step. Nineteen animals were habituated to handling by receiving an injection of saline solution (0.9% NaCl, 1 ml/kg, ip) every day for four days. Then, the animals underwent the protocol described in Luna-Munguia et al. [50]. Briefly, fifteen rats (56 days old) were injected with pilocarpine hydrochloride (340 mg/kg ip; Sigma-Aldrich) 20 min after receiving atropine sulfate (5 mg/kg ip; Sigma-Aldrich). Five to ten minutes after administering the proconvulsive, the animals exhibited head nodding and myoclonic jerks that evolved into recurrent generalized seizures within 40 to 45 min. Rats that did not develop status epilepticus within this time frame, received an additional dose of pilocarpine hydrochloride (170 mg/kg ip). Diazepam (10 mg/kg ip; PiSA) was injected after 90 min of status epilepticus. Control animals (n = 4) received a single injection of atropine sulfate (5 mg/kg ip; Sigma-Aldrich) 20 min prior to vehicle (0.9% saline solution ip); diazepam (10 mg/kg ip) was injected 2 h after the saline injection. A total of 5 rats died during the status epilepticus.

Pilocarpine-treated animals were not video-monitored after the induction of status epilepticus. Therefore, we were not able to identify any spontaneous seizure. However, based on our previous experience using this chronic epilepsy model [50–53], we know that the animals experience the first cluster of spontaneous seizures between day 8 and day 12 post-status epilepticus induction.

Control (n = 4) and status epilepticus-induced rats (n = 10) were scanned at five time points: prior to status epilepticus (50 days old) and 1, 7, 14, and 21 days postinduction. Animals were perfused the day after the last scan. The iron deposits were detected by Perls’ and DAB staining (see below).

Brain Extraction and Histology

Rats were overdosed using an intraperitoneal injection of sodium pentobarbital (Pet’s Pharma). Then, they were intracardially perfused with 0.9% NaCl solution followed by a 4% paraformaldehyde (PFA) solution (pH 7.4). Brains were removed from the skull and postfixed in fresh 4% PFA solution for 24 h at 4 °C. Then, they were immersed in 20% and 30% sucrose solutions at 4 °C. Each specimen was carefully frozen using dry ice and stored at −72 °C until slicing.

The stereotactically injected iron was detected by Perls’ staining. The protocol was partially based on Akyuz et al. [9]. Briefly, brains were sectioned in the coronal plane (40 µm) using a Leica Biosystems 3050S cryostat. The series of sections were preserved in PBS at 4 °C until the mounting and staining day. The 40-µm-thick slices were incubated for 15 min in an equal-volume solution containing 0.2% C₆FeK₄N₆ and 0.2% HCl, both dissolved in deionized water. The sections were counterstained with 0.02% safranine solution. Finally, the sections were mounted on slides and left to dry overnight at room temperature. Images were taken using an AmScope trinocular microscope fitted with a digital camera.

The iron deposits, which were visualized as brain hemosiderin accumulations in the Perls’ + DAB staining of pilocarpine-injected rats, were detected based on Aggarwal et al. [38]. Here, we enhanced the Perls’ reaction using DAB. The brain slices were mounted on slides and rinsed three times in PBS. The slides were submerged for 15 min in an equal-volume solution containing 0.5% C₆FeK₄N₆ and 0.5% HCl. Another PBS wash was done, and the slides were incubated for 10 min in DAB solution (0.5 mg/ml dissolved in 0.1 M PBS). The DAB reaction was triggered with 0.03% H₂O₂ for 15 min. Finally, the slides were washed several times with PBS, dried, and cover-slipped. Images were taken using an AmScope trinocular microscope fitted with a digital camera.

Statistical Analysis

The total susceptibility of the iron deposits was evaluated over time in each brain region where the accumulations were detected. Values are expressed as mean ± S.E.M. Results were analyzed by a one-way ANOVA, and a post hoc Tukey multiple comparisons test was conducted. In all statistical comparisons, significance was assumed at the level of p < 0.05.

Results

QSM as an Effective Tool for Visualizing Iron Deposits In Vivo

We injected four different FeCl₃ solutions (from 0.1 to 100 mM) into the ventral hippocampus of four rats as a first attempt to validate our QSM protocol as an effective technique to detect cerebral iron deposits in vivo. The animal receiving the highest dose of FeCl₃ (100 mM) had a couple of generalized seizures 60 min after surgery. The acquired QSM images showed evident dark holes with a surrounding hyperintensity in the exact place where the FeCl₃ solutions were injected. The size of the holes increased according to the concentration of the injected FeCl₃ solutions (Fig. 2A). To obtain a precise anatomical localization of the iron deposits, we used the T2-weighted TurboRARE structural images. The presence of iron was evident in each of the four injected rats (Fig. 2B). Additionally, the Perls’-stained sections revealed considerable damage to the ventral hippocampus of the animal injected with the highest concentration of FeCl₃ (data not shown). In the other three rats, an intense blue spot was observed at the site of injection. Figure 2C shows the Perls’ staining section of the animal that received the intrahippocampal injection of FeCl₃ 10 mM. Neither imaging nor histological effects were observed in the PBS-injected rat (data not shown).

Fig. 2.

QSM and Perls’ staining to visualize the injected FeCl₃ solutions. To validate our QSM protocol, four different concentrations (0.1, 1, 10, and 100 mM) were injected into the ventral hippocampus of four rats. The red boxes show the locations of iron deposits detected in vivo (dark holes with a surrounding hyperintensity that increased according to the injected concentration) (A). The coronal view of the T2-weighted TurboRARE structural images was used for a precise anatomical localization of the iron deposits (B). Perls’ staining confirmed the place of injection. The blue spot correlates with the dark hole-hyperintensity seen in QSM. Here, we show only the picture of the rat that was injected with the 10 mM FeCl₃ solution (C)

Longitudinal Changes in Cerebral Iron Accumulation After Pilocarpine-Induced Status Epilepticus

QSM images obtained during the basal scan did not show any cerebral iron accumulation (Fig. 3A). Interestingly, all animals had iron deposits (unilateral or bilateral) in the first scan post-status epilepticus induction. The accumulations were detected in specific brain regions, such as the caudate putamen, dorsal hippocampus, thalamus, and ventral hippocampus (Fig. 3B). Some animals also showed them in the primary somatosensory cortex (data not shown). When evaluating the total susceptibility of the deposits during the following weeks, we observed that those localized in dorsal hippocampus, thalamus, and ventral hippocampus increased (Figs. 3C to E and 4A), achieving significance only in the ventral hippocampus on the third and fourth scan post-status epilepticus induction (^*p < 0.05 and ^**p < 0.01, respectively) (Fig. 4B). The reverse effect occurred in caudate putamen (Fig. 4B). The observation of Perls’ + DAB-stained sections confirmed the abnormal presence of the iron deposits detected by QSM (Fig. 4C). No imaging effect was detected in the control animals (Supplementary Figure S1).

Fig. 3.

QSM to detect cerebral iron deposits. None of the animals had cerebral iron deposits during the basal scans (A). However, all animals submitted to pilocarpine-induced status epilepticus showed iron accumulations in the caudate putamen (C. Putamen), dorsal hippocampus (hipp.), thalamus, and ventral hippocampus (hipp.) 24 h postinduction (B). The noninvasive imaging technique allowed us to evaluate the dynamics of the iron deposits weekly (C–E). The small red boxes show the exact place where these accumulations were detected in vivo. The big red boxes show a zoom in of these sites. The coronal view of the T2-weighted TurboRARE structural images was used for a precise anatomical localization of the iron deposits (F)

Fig. 4.

Temporal changes in the cerebral iron deposits. The total susceptibility of the deposits was evaluated at four different time points (1, 7, 14, and 21 days post-status epilepticus induction) in the caudate putamen (C. Putamen), dorsal hippocampus (hipp.), thalamus, and ventral hippocampus (hipp.) A Significant total susceptibility (ppm·mm³) differences were only observed in the ventral hippocampus during the third and fourth scans. In contrast to the other three structures, the total susceptibility of the caudate putamen deposits tended to decrease over time. Data were analyzed by a one-way ANOVA followed by a post hoc Tukey test. Graphs represent the mean ± SEM. ^*p < 0.05 and ^**p < 0.01 (B). The Perls’ + DAB-stained sections confirmed the presence of the iron deposits detected by QSM (C)

Discussion

Iron accumulations and increased ferroptosis markers (ferritin, 4-HNE, and MDA) have been described in the epileptic focus resected from patients with refractory temporal lobe epilepsy [41]. Recent studies in animal models of epilepsy showed similar results [35, 54, 55]. To date, only Aggarwal et al. [38] have been able to detect iron accumulations in ex vivo samples by using QSM. Other reports have even described that treatments focused on reducing ferroptosis clearly reduced seizure severity [35, 56–58]. Based on these references, it seems that epileptic seizures can stimulate ferroptosis, but it is still unknown how iron deposits can induce epilepsy. In this study, we describe the dynamics of brain iron accumulations in a rat model of induced status epilepticus. The use of a noninvasive technique, such as QSM, allowed us to monitor these accumulations longitudinally.

Recently, Auzmendi and Lazarowski [21] suggested that ferroptosis is not only a seizure-induced consequence but also a cause of epilepsy development, a position mainly based on previous clinical and experimental studies [59–61]. In this context, brain iron accumulation could play a central role. In our study, we show evidence of iron deposits present from the first QSM scan post-status epilepticus in the hippocampus, caudate putamen, frontal cortex, and piriform cortex. These are brain regions in which blood–brain barrier integrity is immediately compromised after a severe cerebral insult such as status epilepticus [62]. Therefore, we suggest that the observed iron accumulations in our animals could result from the status epilepticus and may be another factor contributing to future spontaneous recurrent seizures. This idea is consistent with a recent study in a status epilepticus–induced model, in which rats pretreated with CoQ10 and valproic acid exhibited an evident decrease in neuronal death and ferroptosis markers [63]. Moreover, diverse clinical studies have reported that complete removal of hemosiderin during cavernoma surgery is crucial for achieving seizure freedom in patients [64–66].

The iron deposits detected in the dorsal hippocampus of our status epilepticus–induced animals caught our attention. This phenomenon prompted us to consider that an early hippocampal accumulation of iron can precede and act in parallel to widely accepted epileptogenic processes such as hilar cell death, aberrant sprouting, and neuroinflammation. In the specific case of the abnormal growth of mossy fibers and its connection to neuronal death in the hilus and CA3, several experimental studies have demonstrated that their extension depends on the duration of the induced status epilepticus. When this cerebral insult lasts more than one hour, a 90% neuronal death rate is observed, and the onset of spontaneous seizures occurs between 7 and 10 days postinduction; mossy fiber sprouting becomes detectable between day 3 and day 7 [67–69]. Longo and Mello [70] describe that triggering a new ictal event can increase sprouting. These descriptions suggest that early hippocampal iron depositions are not a consequence of the aberrant sprouting or the spontaneous recurrent seizures. However, the progressive increase of the deposits’ size suggests that ongoing seizure activity can be involved in this process.

Our results clearly show cerebral iron deposits; however, we do not know their origins. Diverse experimental studies have reported brain microbleeds as a consequence of status epilepticus induction, suggesting that this phenomenon can provide the required iron to induce hemosiderin formation via hemoglobin degradation [71–73]. We do not dismiss the possibility that some of the iron originates this way, yet the progressive accumulation into deposits suggests that more complex processes are involved. Regardless of the origin of the iron deposits in the brain, hemosiderin can be the source of the ferrous iron excess. Moreover, we also have to consider that microglia, like other macrophages, can also engulf large amounts of iron. This phenomenon promotes the secretion of pro-inflammatory interleukins and neuronal death, rising as an epileptogenic mechanism in posthemorrhagic epilepsies [74]. Also, in vitro experiments have shown that lipopolysaccharide (LPS)-treated microglia can alter neuronal iron homeostasis by increasing the synthesis of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), events that lead to an increased neuronal expression of divalent metal transporter 1 (DMT1), ferritin heavy chain 1 (FTH1), and transferrin receptor 1 (TfR1) [75]. Similar results were described by Urrutia and colleagues when using primary cultures of microglia, astrocytes, and neurons treated with IL-6, TNF-α, or LPS [76]. Recent bioinformatic studies have also shown a strong immunological component in the relationship between ferroptosis and epilepsy, suggesting some ferroptosis-related genes as potential markers (TLR4, HIF-1A, and HMOX1) [77–79].

As previously mentioned, cerebral iron deposits observed in our animals could be associated with a disruption of the blood–brain barrier during the ictal and interictal periods [80, 81]. Once the blood–brain barrier is damaged, it starts the extravasation of serum proteins and other blood-derived factors into the brain parenchyma, facilitating the development of spontaneous recurrent seizures, neuronal loss, and astrogliosis [82, 83]. Moreover, barrier leakage also allows iron to enter from other sources, such as iron bound to heme secondary to hemorrhage, iron bound to transferrin or ferritin, and free plasma iron. Under inflammatory conditions, ferritin levels significantly increase as a consequence of free iron sequestration to mitigate oxidative stress. However, when iron bound to ferritin crosses the compromised blood–brain barrier, it can induce a localized iron overload, resulting in neuronal injury and ferroptosis [84]. In line with this, Garcia-Yebenes and colleagues [85] reported that high levels of iron (measured as serum ferritin) are associated with a worse outcome after stroke induction in mice. Yuan and colleagues [86] conducted a two-sample Mendelian randomization study and suggested that elevated levels of iron, ferritin, and transferrin in serum may be associated with an increased risk of developing epilepsy.

Perls’ and DAB staining performed in this study show some fine dots that may correspond to extracellular iron deposits united by a ferritin core. In tissues, ferritin is an intracellular protein able to store nearly 4500 iron atoms per molecule, releasing them when necessary via ferritinophagy. In vitro studies have shown that ferritin contributes to sustained iron homeostasis; it is released by exocytosis due to increased intracellular iron levels [87, 88]. According to one report, mice with reduced mitochondrial ferritin exhibit neuronal ferroptosis and hippocampal epileptic activity, which are attributed to increased intracellular free iron ions and a decreased function of iron-sequestering proteins [89]. On the other hand, another explanation for the dotted marks we observed may be related to iron, microglia, and proinflammatory processes. Recent studies report the involvement of microglia in ferroptosis induced by stroke, ischemia/reperfusion, and amyotrophic lateral sclerosis [90–92]. In this regard, in vitro experiments by Urrutia and colleagues [76] showed that microglial stimulation (with IL-6, TNF-α, or LPS) facilitates hepcidin secretion and DMT1 expression, promoting detrimental iron accumulation in the cells. Other authors recently described that some anti-inflammatory treatments promote physiological iron storage by decreasing ferritin heavy chain expression and increasing ferritin light chain expression [92, 93]. In vitro studies using ferromagnetic nanoparticles have also demonstrated that intracellular nonheme iron accumulation is safe at certain concentrations (approximately 50 µg/ml) [94]. However, higher concentrations promote oxidative stress and cell death [95, 96]. This suggests that cells have a buffer capacity to store a certain excess of iron without impacting viability.

Limitations

It is important to mention some considerations about our study. First, QSM methods are affected by residual background field contributions and artifacts; specifically, near air–tissue interfaces. While these effects are minimized in deep brain structures, which comprise the majority of our findings, it is complicated to detect the cortical iron deposits. Overcoming this challenge will add valuable information to our data when evaluating iron dynamics in animal models. Second, noise and the point-dipole problem also complicate the accuracy of the measured susceptibilities [97]. Third, when quantifying iron deposits, some methodological aspects should be considered. The susceptibility thresholds that we used to detect (0.1 ppm) and segment (0.05 ppm) the deposits are fixed operational values. Moreover, the total susceptibility used to characterize each deposit is a relative measure and cannot provide absolute iron concentrations. Formal threshold optimization and quantitative histological calibration remain as objectives for future work.

Conclusion

Our study provides compelling evidence of iron deposits after status epilepticus induction. Moreover, it proves that QSM is a useful noninvasive technique for detecting these deposits longitudinally. While this study is preclinical in nature, the methods used here are applicable in human scanners. Thus, our findings underscore the potential of using QSM for clinical applications in which cerebral iron accumulation is suspected (posttraumatic, poststroke, postischemic, and postinfection) [98, 99]. Future experiments are crucial to determine the origin of the iron that accumulates in specific brain regions and understand/prevent the vicious cycle that occurs after the insult is triggered (iron deposits—seizures—new iron deposits).

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contribution

FM, AL, LC, JA, and HL-M contributed to the conception, methodology, and design of the study. FM and HL-M performed imaging acquisition and histological procedures. FM and L-VM performed QSM imaging and statistical analyses. LC provided the code and supervised the analysis of iron detection. FM, AL, JA, and HL-M wrote, reviewed, and edited the first manuscript. All authors reviewed the manuscript and approved the submitted version.

Funding

This work was supported by UNAM-DGAPA-PAPIIT (IN211326 for HL-M and IN213423 for LC), PICT2019-01282 for JA, and CONAHCYT (CF-218-2023 for LC).

Data Availability

All data generated and analyzed during this study will be made available by the authors, without undue reservation.

Declarations

Ethics Approval and Consent

Research was approved by the Institute of Neurobiology Ethics Animal Care Committee (protocol 105 A).

Competing interests

The authors declare no competing interests.